The MGMTs (EC 2.1.1.63; also referred to in the art as O.sup.6 -alkylguanine-DNA alkyltransferases) are DNA repair proteins found in most living organisms which transfer alkyl groups from the O-6 position of guanine in alkylated DNA to a cysteine residue of their own polypeptide chains, thereby becoming catalytically inactive. Since each MGMT molecule acts but once, the repair capacity of a living cell is determined by the number of MGMT molecules in a cell at the time of DNA alkylation, and the rate of its resynthesis.
The MGMT levels of living cells are directly correlated with the response of those cells to S.sub.n 2 alkylating agents, such as the clinically useful anti-cancer chemotherapeutic drug group, the chloroethylnitrosoureas. A group of human tumors and cell strains have the MER.sup.- phenotype and are deficient in MGMT activity, and these tumors and cells are extremely sensitive to killing by these drugs.
Various reviews of the MGMTs and their activity have appeared in the literature, including: DANIEL B. YAROSH, "THE ROLE OF O.sup.6 -METHYLGUANINE-DNA METHYLTRANSFERASE IN CELL SURVIVAL, MUTAGENESIS AND CARCINOGENESIS", MUTATION RESEARCH, VOLUME 145, PAGES 1-16, 1985; ANTHONY E. PEGG, "PROPERTIES OF MAMMALIAN O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE, MUTATION RESEARCH, VOLUME 233, PAGES 165-175, 1990; and ANTHONY E. PEGG, "MAMMALIAN O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE: REGULATION AND IMPORTANCE IN RESPONSE TO ALKYLATING, CARCINOGENIC AND THERAPEUTIC AGENTS", CANCER RESEARCH, VOLUME 50, PAGES 6119-6129, 1990, the relevant portions of which are incorporated herein by reference.
Scientists in the field have long recognized that measurement of MGMT levels in human tumors would be an important contribution to improving chloroethylnitrosourea chemotherapy of cancer. For example, M. Watatani, M. Ikenaga, T. Hatanaka, M. Kinuta, S. Takai, T. Mori and S. Kondo predicted in 1985 that "If the MER.sup.- phenotype could be detected by some means in biopsy specimens, it should be possible to cure this type of malignant tumor by appropriate chemotherapy." See "ANALYSIS OF N-METHYL-N'-NITRO-N-NITROSOGUANIDINE (MNNG)-INDUCED DNA DAMAGE IN TUMOR CELL STRAINS FROM JAPANESE PATIENTS AND DEMONSTRATION OF MNNG HYPERSENSITIVITY OF MER.sup.- XENOGRAFTS IN ATHYMIC NUDE MICE", CARCINOGENESIS, VOLUME 6, PAGES 549-553 at 552, 1985.
Similarly, T. Aida, R. Cheitlin and W. Bodell wrote in 1987: "Our results suggest that measurement of O.sup.6 -AT [MGMT] activity in human brain tumor biopsy specimens may be a good prognostic indicator of clinical resistance to BCNU [a chloroethylnitrosourea chemotherapy agent]." See "INHIBITION OF O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY POTENTIATES CYTOTOXICITY AND INDUCTION OF SCEs IN HUMAN GLIOMA CELLS RESISTANT TO 1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA", CARCINOGENESIS, VOLUME 8, PAGES 1219-1223 at 1222, 1987.
Along these same lines, C. Fujio, H. R. Chang, T. Tsujimura, K. Ishizaki, H. Kitamura, and M. Ikenaga concluded in 1989 that "measurement of ATR [MGMT] activity in biopsy specimens may provide a useful guide to predict the response to chemotherapy" and, in particular, that "MER tumors may be efficiently cured by treatment with alkylating agents, including ACNU, provided that we could detect the MER.sup.- tumors at their early stage of development." These authors also point out that about one-fifth of human tumor cell strains established in the United States show a MER.sup.- phenotype and that similarly, a MER.sup.- frequency of 15% has been found in tumor cell lines derived from Japanese patients. See "HYPERSENSITIVITY OF HUMAN TUMOR XENOGRAFTS LACKING O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE TO THE ANTI-TUMOR AGENT 1-(4-AMINO-2-METHYL-5-PYRIMIDINYL) METHYL-3-(2-CHLOROETHYL)-3- NITROSOUREA", CARCINOGENESIS, VOLUME 10, PAGES 351-356 at 351 and 355, 1989.
Prior to the present invention, the most sensitive assay for MGMT was an activity assay, which measured the transfer of alkyl groups from DNA to the MGMT protein. This assay, however, relies on collecting fresh samples, and invariably includes irrelevant cells along with relevant cells in the extract used for assay. For years, it has been recognized in the art that an immunoassay for MGMT levels in single cell preparations would represent an important and essential improvement having great clinical relevance.
Thus, in 1985, Thomas Brent, a leading researcher in this field described the problems with activity measurements and the need for an immunoassay as follows: "In practice, however, due to cellular heterogeneity and necrosis, transferase activity measurements in human tumor biopsies are fraught with difficulty. The development of an antibody for the human alkyltransferase and a cytological immunoassay for the transfer protein in tumor cells should enable these predictive assays to be achieved." See "ISOLATION AND PURIFICATION OF O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE FROM HUMAN LEUKEMIC CELLS. PREVENTION 0F CHLOROETHYLNITROSOUREA-INDUCED CROSS-LINKS BY PURIFIED ENZYME", PHARMACOLOGICAL THERAPEUTICS, VOLUME 31, PAGES 121-139 at 138, 1985.
In view of this clear need for an immunoassay and, in particular, a single cell immunoassay, substantial efforts by a variety of groups throughout the world have been made to develop antibodies to MGMT. Notwithstanding these efforts, prior to the present invention, truly useful and practical antibodies to MGMT, in particular, monoclonal antibodies, have not been found. As discussed below, this failure by the leading groups to satisfy this long felt need has resulted from the approaches they took in selecting immunizing materials, i.e., the groups did not use MGMT having transferase activity as their immunizing material.
Thus, the Bigner/Brent group reported the development of monoclonal antibodies using denatured MGMT, i.e., MGMT which had been purified using polyacrylamide-SDS gels and thus lacked transferase activity. The antibodies were only able to recognize MGMT after denaturation by SDS and were thus unsuitable for use in single cell assays. See M. VON WRONSKI, T. BRENT, C. PEGRAM, D. BIGNER, "MONOCLONAL ANTIBODIES AGAINST HUMAN O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, VOLUME 30, PAGE 486, 1989; and T. BRENT, M. VON WRONSKI, C. PEGRAM AND D. BIGNER, "IMMUNOAFFINITY PURIFICATION OF HUMAN O.sup.6 -METHYLGUANINE-DNA METHYLTRANSFERASE USING NEWLY DEVELOPED MONOCLONAL ANTIBODIES", CANCER RESEARCH, VOLUME 50, PAGES 58-61, 1990. See also, M. VON WRONSKI, D. BIGNER AND T. BRENT, "EXPRESSION OF O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE IN MER.sup.+ AND MER.sup.- HUMAN CELL EXTRACTS PROBED WITH SPECIFIC MONOCLONAL ANTIBODIES", CANCER COMMUNICATIONS, VOLUME 1, PAGES 323-327, 1989.
Around this same time period, the present inventor also attempted to develop monoclonal antibodies to MGMT using materials which had been partially purified and denatured on glass filters. See D. YAROSH AND J. CECCOLI, "MONOCLONAL ANTIBODIES AGAINST THE HUMAN O.sup.6 -METHYLGUANINE-DNA METHYLTRANSFERASE", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, VOLUME 29, PAGE 1, 1988. Although mouse serum containing the antibodies would recognize MGMT on Western blots, i.e., the antibodies would recognize the denatured protein, they did not recognize the native material. Also, no worthwhile hybridomas were obtained from this work, since the antibodies which were produced had low affinity and were generally not useable.
After their work with denatured MGMT, the Bigner/Brent group turned to the use of synthetic peptide fragments corresponding to different portions of the amino acid sequence of human MGMT to produce polyclonal antibodies to MGMT. Specifically, they used three fragments corresponding to predicted hydrophilic portions of the MGMT molecule and a fourth fragment centered around the cysteine residue which accepts the alkyl group from alkylated DNA. L. OSTROWSKI, C. PEGRAM, M. VON WRONSKI, P. HUMPHREY, X. HE, S. SHIOTA, S. MITRA, T. BRENT AND D. BIGNER, "PRODUCTION AND CHARACTERIZATION OF ANTIPEPTIDE ANTIBODIES AGAINST HUMAN O.sup.6 -METHYLGUANINE-DNA METHYLTRANSFERASE", CANCER RESEARCH VOLUME 51, PAGES 3339-3344, 1991.
The best that can be said for this work is that it achieved mixed results. Thus, all four polyclonal antibodies were able to precipitate inactive (methylated) MGMT, two of the four recognized MGMT on Western blots, but the other two did not, and only one of the four would precipitate native MGMT. No evidence is presented in the Ostrowski et al. paper that any of the antibodies could be used for single cell staining. Moreover, in terms of large scale, commercial screening of tumor samples, polyclonal antibodies are impractical since individual animals have different immunological responses to a given antigen.
Similar results with polyclonal antibodies raised against synthetic peptide fragments were obtained by Anthony E. Pegg, another leading scientist in the field, and his group. A. E. PEGG, L. WIEST, C. MUMMERT, AND M. E. DOLAN, "PRODUCTION OF ANTIBODIES TO PEPTIDE SEQUENCES PRESENT IN HUMAN O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE AND THEIR USE TO DETECT THIS PROTEIN IN CELL EXTRACTS", CARCINOGENESIS, VOLUME 12, NUMBER 9, PAGES 1671-1677, 1991; and A. E. PEGG, L. WIEST, C. MUMMERT, L. STINE, R. C. MOSCHEL, AND M. E. DOLAN, "USE OF ANTIBODIES TO HUMAN O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE TO STUDY THE CONTENT OF THIS PROTEIN IN CELLS TREATED WITH O.sup.6 -BENZYLGUANINE OR N-METHYL-N'-NITRO-N-NITROSO-GUANIDINE", CARCINOGENESIS, VOLUME 12, NUMBER 9, PAGES 1679-1683, 1991. In this case, six antisera were prepared and all were found to react with MGMT on Western blots. However, none of the antisera "showed any detectable interaction with the native protein either before or after affinity purification" which Pegg et al. describe as "quite surprising" since relatively hydrophilic sequences were used for the immunization. CARCINOGENESIS at 1674 and 1677. Pegg et al. describe the sum and substance of their work as follows: "[T]he development of sensitive and specific immunoassays that can be used to determine the occurrence and intracellular distribution of the alkyltransferase requires the development of more specific reagents. It is possible, but at present unproven, that this may be accomplished by more extensive immunopurification of the present antibodies or by modifying the peptide sequences used for immunization." CARCINOGENESIS at 1677.
The polyclonal antibody approach, in this case raised against recombinant human MGMT, is also described in an abstract published in March 1992 by S. M. LEE, J. RAFFERTY, C. Y. FAN, N. THATCHER, AND G. P. MARGISON, "REGIONAL AND CELLULAR HETEROGENEITY OF EXPRESSION OF O.sup.6 -ALKYLGUANINE-DNA ALKYLTRANSFERASE IN MELANOMA", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, VOLUME 33, MARCH 1992, PAGE 547.
The overall current state of the art was aptly summarized in a review article by Dr. Pegg which recently appeared in the FEDERATION OF AMERICAN SOCIETIES OF EXPERIMENTAL BIOLOGY JOURNAL, VOLUME 6, PAGES 2302-2310 at 2306: "It is apparent that much more work in this area is needed and that there is an urgent need for a method for studying the distribution of alkyltransferase on a cellular basis. This measurement is of major importance because of the role of the alkyltransferase in conferring resistance to chemotherapeutic agents . . . The obvious possibility in this regard is immunohistochemistry, but it remains to be demonstrated that antisera of sufficient specificity and affinity to measure the small amounts of alkyltransferase present in cells can be prepared."
It is to this challenge that the present invention is directed and as demonstrated below, the invention achieves the goal of providing monoclonal antibodies which have sufficient specificity and affinity to detect MGMT in single cell preparations.